Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.     

The prevalence and characterization of verotoxin-producing Escherichia coli isolated from cattle and pigs in an abattoir in Hong Kong

The aim of the study was to define the prevalence of verotoxin-producing Escherichia coli (VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin (VT) by PCR and cytotoxicity assays. VTEC was isolated from 415 and 1-8% of cattle faecal and carcass samples and from 2.1 and 0.2% of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157:H7 and eaeA+. The most prevalent vt genotype among bovine VTEC was vtl+vt2 (73.8%) and in porcine VTEC was vt2e+ (30%). None of the porcine VTEC isolates and 9.3% of the bovine VTEC isolates was eaeA+. The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef), the low VTEC carriage in pigs, the rarity of additional virulence factors (caeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eacA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.     

A prospective study of exposure to verotoxin-producing Escherichia coli among Canadian children with haemolytic uraemic syndrome. The CPKDRC co-investigators.

Haemolytic uraemic syndrome (HUS) is a leading cause of acute renal failure in childhood. Although infection with Escherichia coli O 157. H7 has been associated with HUS in North America and Europe, only a limited number of studies have examined the role of other verotoxin-producing E. coli (VTEC) serotypes in this condition. To address this issue, we conducted a comprehensive, prospective microbiological study of patients treated for HUS at eight Canadian hospitals in the summer of 1990. Of the 34 consecutive patients with HUS enrolled over 4 months, E. coli O 157. H7 was isolated from the stools of 26, and other E. coli serotypes were isolated from four patients. In four subjects no pathogenic E. coli serotypes were identified on stool culture. Using oligonucleotide probes specific for VT-1 and VT-2, verotoxin genes were detected in the stool isolates of all patients with E. coli O 157. H7, and from two with other E. coli serotypes. Two other patients had at least a fourfold rise in anti-verotoxin antibodies. Strong evidence of exposure to a verotoxin was present in 30/34 (88%). Patients with E. coli O 157. H7 infection were more likely to develop an antibody response to VT-2 than to VT-1 (22/22 vs 12/22; P = 0.002). These results further strengthen the association of HUS with verotoxin-producing E. coli in North America, and confirm that E. coli serotypes other than O 157.H7 are isolated in a small proportion of summertime HUS episodes.     

Characterization of verocytotoxin-producing Escherichia coli O157 isolates from patients with haemolytic uraemic syndrome in Western Europe.

Fifty verocytotoxin (VT)-producing Escherichia coli (VTEC) strains of serogroup O157 were characterized by phage typing, polymerase chain reaction (PCR) for VT genes and the E. coli attaching and effacing (eae) gene, and random amplified polymorphic DNA-PCR (RAPD-PCR) fingerprinting. The collection represented isolates obtained from patients with diarrhoea-associated haemolytic-uraemic syndrome (D+ HUS) and their family contacts, isolated in the Netherlands, Belgium and Germany between 1989 and 1993. Based on isolates from separate families (n = 27) seven different phage types were identified, types 2 (44%) and 4 (33%) were predominant. Eighty-five percent of the strains contained only VT2 gene sequences and 15% both VT1 and VT2. All strains of the dominant phage types 2 and 4 carried the VT2 gene. Strains that belonged to the minor phage types 8, 14, 32 carried both VT1 and VT2 genes, with the exception of two isolates identified as phage types 49 and 54 which contained only VT2 genes. All O157 VTEC strains possessed the chromosomally-located eae gene, which indicates its usefulness as virulence marker. RAPD-PCR fingerprinting identified four distinct banding patterns, with one profile found among 79% of the strains. Based on the combined results of all typing methods used in this study, the collection of 50 O157 VTEC strains could be divided into nine distinct groups. Strains isolated from different persons within one family could not be distinguished by any of these methods. The data suggest that O157 VTEC strains are members of one clone that has become widely distributed.     

Persistence of Escherichia coli O157:H7 in dairy cattle and the dairy farm environment.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.     

A case-control study of sporadic infection with O157 and non-O157 verocytotoxin-producing Escherichia coli.

Potential risk factors for sporadic verocytotoxin-producing Escherichia coli (VTEC) infection in Belgium were investigated in a matched case-control study. Thirty-seven cases, 8 infected with O157 VTEC strains (all eaeA-positive), 29 with non-O157 VTEC strains (13 eaeA-positive and 16 eaeA-negative) and 69 matched controls were interviewed. In a conditional logistic regression analysis, consumption of fish appeared to be a risk factor for infection (adjusted odds ratio (OR) 3.25, P = 0.04). Contact with dogs (OR 0.27, P = 0.04) and consumption of shellfish (OR 0.19, P = 0.05) showed a negative association, corresponding to a decrease in risk. These findings might be explained if low level environmental exposure to VTEC induces protective immunity. Eating raw meat, a frequent habit in Belgium, or hamburgers, or eating in a fast-food restaurant was not more frequently reported by cases than controls. The exposures causing sporadic infections with VTEC, in particular non-O157 strains, may be very different from those which led to outbreaks, and may account for more cases overall.     

2011-2019年33株人源非O157产志贺毒素大肠埃希菌耐药表型及耐药相关基因分析

目的了解人源非O157产志贺毒素大肠埃希菌(STEC)抗生素耐药表型及耐药基因携带状况。方法33株非O157 STEC菌株来自2011-2019年7个省份,包括青海、黑龙江(各1例),广西、山东(各2例),广东(4例),河南(11例),上海(12例),均分离自感染性腹泻患者粪便标本。采用改良微量肉汤法对非O157 STEC进行19种抗生素最小抑菌浓度(MIC)检测;通过全基因组测序进行O∶H血清分型、多位点序列分型(MLST)及耐药相关基因预测。结果33株非O157 STEC分为19种O∶H血清型、17种MLST序列型;10株菌对1种或以上抗生素耐药,5株为多重耐药菌株,对四环素耐药率最高(30.3%,10株),并检出阿奇霉素耐药菌株(12.1%,4株),但所有菌株均对碳青霉烯类药物敏感;共检出22种耐药基因,全部菌株均携带blaEC基因,并检出超广谱β-内酰胺酶(ESBL)基因型blaCTX-M-15(3.0%,1株),首次检出fosA7基因。结论中国7省份33株人源非O157 STEC中存在多重耐药和阿奇霉素耐药菌株,且携带多种耐药基因型,但均对碳青霉烯类药物敏感。     

Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers.

The distribution of the Escherichia coli attaching and effacing (eae) gene in strains of verotoxin-producing E. coli (VTEC) isolated from cattle and humans was studied. The majority of strains isolated from humans with bloody diarrhoea or HUS and cattle with severe diarrhoea were eae positive (82 and 83% respectively). In contrast, 59% of VTEC isolated from asymptomatic cattle were eae negative and of the remaining 41% that were eae positive, the majority were serotype O157. H7. The nucleotide sequence of the 3'' end of the eae gene of enteropathogenic E. coli (EPEC) of serotype O55. H7 was found to be almost identical to that of serotype O157. H7. Specific primers are described which detect the eae sequences of VTEC serotypes O157. H7, O157. H-, and EPEC serotypes O55. H7 and O55. H-. The nucleotide sequence of the 3'' end of the eae gene of serotype O111. H8 differed significantly from that of O157. H7. Primers were developed to specifically identify the eae sequences of VTEC serotypes O111. H- and O111. H8. We conclude that whereas the majority of VTEC associated with disease in cattle and humans possess the eae gene, the gene itself may not be necessary to produce haemorrhagic colitis and HUS. Sequence heterogeneity in the 3'' end of eae alleles of VTEC permits specific identification of subsets of these organisms.     

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.     

Verocytotoxin-producing Escherichia coli in foodstuffs of animal origin

While much research effort has been targeted at the verocytotoxin-producing Escherichia coli (VTEC) serotype O157:H7, it is becoming more evident that other VTEC serotypes can also be associated with human foodborne disease. An increasing number of these non-O157 serotypes have been isolated from food sources and from humans suffering from haemolytic-uraemic syndrome and diarrhoea. The aim of our work was to investigate the prevalence of VTEC O157 and non-O157 in foodstuffs of animal origin using two rapid enzymatic procedures. Various types of food samples, 352 in total, were tested: 233 with the Premier EHEC, a screening test which directly detects the presence of verocytotoxin, regardless of serotype, while 119 of these with the Vidas ECO, which is a specific screening test for E. coli O157:H7, together with the Premier EHEC. Two samples were positive for VTEC, one of serogroup O126 and the other was non-serotypable. Another sample was positive in the test specific for E. coli O157:H7, but was not confirmed by culture. This study suggests that VTEC strains are not prevalent in Italy, and that the isolation of serogroup O157 is relatively infrequent. This leads us to conclude that there is little chance of exposure to pathogen for the average consumer in Italy.     

Human infections with non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009

We characterized 97 non-O157 Shiga toxin (stx)-producing Escherichia coli strains isolated from human patients during 2000-2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H-, O103:H2, O121:H19, and O145:H28/H-) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H- was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to ≥1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx-producing E. coli infections in Switzerland most often occurred as single cases.     

Prevalence of the eaeA gene in verotoxigenic Escherichia coli strains from dairy cattle in Southwest Ontario.

This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA gene showed that 151 (35.2%) strains were positive for the eaeA gene. All isolates (9-21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7-34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA-negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle.     

Genotyping and antimicrobial resistance patterns of Escherichia coli O157 originating from cattle farms

During a Escherichia coli O157 prevalence study on cattle farms, 324 E. coli O157 isolates were collected from 68 out of 180 cattle farms. All isolates harbored the eaeA gene and the enterohemolysin (ehxA) gene. The majority of the strains only contained vtx2 (245 isolates), the combination of vtx1 and vtx2 was detected in 50 isolates, and in 29 isolates none of the vtx genes was present. Pulsed-field gel electrophoresis (PFGE) revealed that at a similarity level of 98% the isolates grouped into 83 different genotypes, 76 of which were only detected on one farm. Twenty-two out of the 68 positive farms harbored isolates belonging to more than one PFGE type, with a maximum of four different PFGE types. Minimal inhibitory concentrations of 10 antimicrobial agents were determined on a subset of 116 isolates, that is, one isolate per positive age category per farm. Acquired resistance to at least one antimicrobial agent was detected in 18 isolates and within a farm, only one resistance pattern was observed. All these 18 isolates were resistant toward streptomycin, and 16 of them also showed resistance toward sulfisoxazole. Six isolates were resistant to three or more antimicrobial agents.     

Serum antibodies to secreted proteins in patients infected with Escherichia coli O157 and other VTEC.

Certain strains of verotoxigenic Escherichia coli (VTEC), and in particular those belonging to serogroup O157, cause attaching and effacing (AE) lesions of the host gut mucosa during pathogenesis. The mechanisms involved with bacterial attachment and the destruction of microvilli are determined by a cluster of genes within the LEE region, which also encode five secreted proteins. Sera from patients with antibodies to the lipopolysaccharide (LPS) of E. coli O157 and other VTEC were tested for antibodies to these secreted proteins. Twenty-one of 34 (62%) sera with antibodies to the lipopolysaccharide (LPS) of E. coli O157 also contained antibodies to one or more of the secreted proteins. Five of 12 sera containing antibodies to the LPS of a range of other VTEC serogroups also contained antibodies to 1 or more of the 5 secreted proteins, as did 16 of 70 (23%) sera from patients with haemolytic uraemic syndrome (HUS), haemorrhagic colitis (HC) or diarrhoea, but without bacteriological evidence of infection with VTEC and which did not contain antibodies to VTEC serogroups O5, O115, O145, O153 or O157. The detection of serum antibodies to secreted proteins may provide additional information for interpreting the results of established lipopolysaccharide-based VTEC serology.     

Prevalence and characteristics of Escherichia coli serotype O157:H7 and other verotoxin-producing E. coli in healthy cattle.

From February to July of 1994, 328 faecal samples from 32 herds were collected and verotoxin-producing Escherichia coli (VTEC) found on 84% of the farms. The proportion of animals infected varied from 0-63%. VTEC were recovered from 52 (20%) of 257 cows and from 16 (23%) of 71 calves. Although the VTEC belonged to 25 different serogroups, 7 (O8, O20, O22, O77, O113, O126 and O162) accounted for 46% of strains. Nearly 45% of the strains. Nearly 45% of the 83 bovine VTEC strains belonged to serogroups associated with haemorrhagic colitis and haemolytic uraemic syndrome in humans. However, only 2 (2%) of 83 VTEC strains isolated from cattle belonged to enterohaemorrhagic E. coli (EHEC) serotypes (O26:H11 and O157:H7), and only 8 (10%) were positive for the attaching and effacing E. coli (eae) gene sequence. Polymerase chain reaction (PCR) showed that 17 (20%) of VTEC strains carried VT1 genes, 43 (52%) possessed VT2 genes, and 23 (28%) carried both VT1 and VT2 genes. Characterization of VTEC isolates revelated a heterogeneous population in terms of serogroup and toxin type in the positive herds. This study confirms that healthy cattle are a reservoir of VTEC, but, the absence of eae genes in most bovine VTEC strains suggests that they may be less virulent for humans than eae-positive EHEC.     

Verocytotoxigenic (Shiga toxin-producing) Escherichia coli: virulence factors and pathogenicity in the farm to fork paradigm

Verocytotoxigenic Escherichia coli (VTEC) are a good example of the evolution and emergence of pathogenic E. coli. Unknown before the late 1970s, these bacteria are a major cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. The production of verocytotoxins is the main virulence feature of VTEC but cannot be solely responsible for full pathogenicity. VTEC associated with severe human disease are usually capable of colonizing the intestinal mucosa with a characteristic attaching-and-effacing mechanism, genetically governed by the locus of enterocyte effacement, and possess other mobile genetic elements carrying additional virulence genes such as plasmids, phages, and pathogenicity islands (e.g., O-I 122). Despite the huge amount of data collected after the sequencing of the full genome of VTEC O157, the virulence and the evolution of the different VTEC serotypes have only been partially unraveled. A greater understanding of the factors governing the development of severe disease in humans and the colonization of animal hosts must be achieved before effective intervention strategies aimed at the reduction of the burden of infection can be developed. Defining all the factors characterizing a fully pathogenic VTEC strain will be crucial to improve the efficacy of the diagnosis of human infections, the surveillance of animal reservoirs, the assessment of public health risks, and the development of control interventions. An overview of the VTEC virulence factors, including their genetic basis and function, would start this process and is the objective of this article.     

Sporadic isolates of Escherichia coli O157.H7 investigated by pyrolysis mass spectrometry.

Thirty-six encoded isolates of Escherichia coli. 32 of which were of serotype O157, were examined by pyrolysis mass spectrometry (PyMS). Thirty-one of the serotype O157 isolates possessed the flagellar antigen H7 and produced Verocytotoxin (VT), the other isolate serotyped as H45 and was non-toxigenic. Eighteen of the VT-producing E. coli (VTEC) isolates were from sporadic disease in residents of the Northern Region. Standard principal component (PC) and canonical variate (CV) analysis of the data distinguished only the four non-O157 isolates from the remainder which were indistinguishable by this approach. A similarity matrix based on differences between individual CV means distinguished a further ten isolates. The matrix correctly clustered 2 pairs of isolates from siblings and 4 isolates from an affected family. A further 5 clusters of 3 or more isolates and 6 pairs of isolates were defined. These groupings proved to be homogenous for toxin phenotype but occasionally entrained isolates of dissimilar phage type. However, in general, PyMS-derived clustering of apparently sporadic isolates accorded with geographical locations as determined by postcode. PyMS, which is a quick and high volume capacity phenotypic technique, may be a useful addition to existing methods in the investigation of the epidemiology of sporadic VTEC disease.     

Laboratory-confirmed non-O157 Shiga toxin-producing Escherichia coli--Connecticut, 2000-2005

Shiga toxin-producing Escherichia coli (STEC) infection causes diarrhea that is often bloody and can result in potentially life-threatening hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 is the most common cause of STEC infection in the United States, producing 73,000 illnesses annually, according to the last estimate in 1999. Unlike O157, however, little is known about the incidence of non-O157 strains. Because STEC other than O157 are not commonly identified, the incidence, trends, and epidemiology of non-O157 STEC are not well understood. To assess trends in Shiga toxin enzyme immunoassay (Stx EIA) testing by local clinical laboratories, the Connecticut Department of Public Health (CTDPH) analyzed results of confirmatory testing conducted in the state laboratory during 2000--2005. The findings indicated that a total of 403 STEC infections were reported by clinical laboratories in Connecticut, including 207 identified as STEC by Stx EIA testing alone, and that the use of Stx EIA increased from 2000 to 2005. Use of Stx EIA without prompt culture confirmation can delay or prevent serotyping and subtyping of isolates and detection of both O157 and non-O157 STEC outbreaks. Public health authorities in all states should ensure that clinical laboratories forward Stx EIA-positive specimens to the state laboratory for isolation and identification of STEC, as recommended by the Association of Public Health Laboratories and CDC.     

Applicability of a multiplex PCR to detect the seven major Shiga toxin-producing Escherichia coli based on genes that code for serogroup-specific O-antigens and major virulence factors in cattle feces

An 11-gene multiplex polymerase chain reaction (mPCR) was developed based on genes that code for serogroup-specific O-antigens and four major virulence factors (intimin, enterohemorrhagic hemolysin, and Shiga toxins [Stx] 1 and 2), to detect O157 and the "top six" non-O157 (O26, O45, O103, O111, O121, and O145) Shiga toxin-producing Escherichia coli (STEC). The assay specificity was validated with pure cultures of seven major STEC (185 strains), 26 other STEC (65 strains), non-STEC (five strains), and 33 strains of other genera and species. Sensitivity of the assay with cattle fecal sample spiked with pooled cultures of seven major STEC was 10(5) colony-forming units (CFU)/g before enrichment and 10(2) CFU/g after enrichment. The applicability of the assay to detect STEC in fecal samples (n=50), before and after enrichment, was evaluated by comparing with culture-based methods for O26, O111, and O157. The mPCR assay of 50 fecal samples showed seven (14%) positive before enrichment and 23 (46%) positive after enrichment for one or more of the seven O-groups. Overall, 17 isolates from 17 fecal samples and 27 isolates (four for O26, three for O45, and 20 for O103) from 19 fecal samples were obtained, by culture-based methods, for O157 and non-O157 serogroups, respectively. None of the 27 non-O157 isolates possessed the stx genes, suggesting that cattle harbor Shiga toxin-negative E. coli belonging to the "top six" non-O157 serogroups. Our data, although based on a limited number of samples, suggest that the sensitivities of the mPCR and culture-based methods in detecting the seven serogroups of STEC in feces differed between O-groups. An obvious limitation of our mPCR is that the concurrent detection of virulence genes and the serogroups in a sample does not necessarily associate the virulence genes with the prevalent serogroups in the same sample. The major application of our 11-gene mPCR assay may be in identifying putative colonies of STEC obtained by culture-based methods.     

Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli

Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).